Technical Process for Preparation of Genistein

Article abstract of DOI:10.1021/acs.oprd.5b00425

Development and scale-up of the synthetic process for genistein preparation are described. The process was designed with consideration for environmental and economical aspects and optimized in a laboratory scale. In a scale up, on every step quantity of the environmentally unfriendly substrates or solvents was reduced without compromising the quality of the final product, and the waste load was significantly diminished. The optimal duration times of the individual stages were determined, and the number of operations was reduced, leading to lowering of energy consumption. Elaborated process secures good yield and quality expected for pharmaceutical substances.

Full text of DOI:10.1021/acs.oprd.5b00425

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Communication
Technical process for preparation of genistein
Katarzyna Filip, Ewa Kleczkowska-Plichta, Zbigniew Ara#ny, Grzegorz
Grynkiewicz, Magdalena Polowczyk, Krzysztof Gabarski, and Kinga Trzci#ska
Org. Process Res. Dev., Just Accepted Manuscript • DOI: 10.1021/acs.oprd.5b00425 • Publication Date (Web): 03 Jun 2016
Downloaded from http://pubs.acs.org on June 4, 2016
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Title: Technical process for preparation of genistein
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Authors:
Katarzyna Filip*, Ewa KleczkowskaꢀPlichta, Zbigniew Araźny, Grzegorz Grynkiewicz
Magdalena Polowczyk, Krzysztof Gabarski, Kinga Trzcińska
*Corresponding author, k.filip@ifarm.eu
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Affiliations
Pharmaceutical Research Institute, Rydygiera 8, 01ꢀ793 Warsaw, Poland
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Table of Content
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Abstract
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Development and scaleꢀup of synthetic process for genistein preparation is described. The
process was designed with consideration for environmental and economical aspects and
optimised in a laboratory scale. In a scale up, on every step quantity of the environmentally
unfriendly substrates or solvents was reduced without compromising the quality of the final
product and waste load was significantly diminished. The optimal duration times of the
individual stages were determined and the number of operations was reduced, leading to
lowering of energy consumption. Elaborated process secures good yield, and quality expected
for pharmaceutical substances.
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Keywords
Genistein synthesis, scale up, technical process
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INTRODUCTION
Like many other biologically active natural products, isoflavone genistein (
1
, Figure 1)
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is undergoing a lengthy process of image transformations, from scientific curiosity, through
biochemical molecular probe, to a candidate for chemopreventive agent or even multitarget
therapeutic1ꢀ3. These changes imply inevitable switch of the chemical entity status from a
reagent to an industrial material (specialty chemical) category, with regulatory requirements
such as source identification, process and methods validation, and batch certification.
Pharmaceutical fine chemicals, including active pharmaceutical ingredients (API),
constitute a significant global market fraction, estimated at 8 – 9% of the total pharmaceutical
sales at the manufacturer level (presently estimated at $ 80 billion)4. While popular large
volume of API product manufacturing tends to get consolidated and automated, great number
of small scale APIs and intermediates are presently at a stage of testing manufacturing
capabilities between synthetic variants and biotechnological alternatives, at process
development groups of R&D oriented enterprises4. The latter applies in particular to selection
of natural products which have already found an applications in therapy.
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Figure 1 Chemical structure of genistein (compound 1)
Genistein 1, an isoflavone found in many Fabaceae plants and important nonꢀ
nutritional constituent of soy beans (Glycine max Merill) is a well known plant metabolite
from phenylpropanoid pathway, chiefly because of its presence in numerous phytoestrogenic
dietary supplements5. In fact, the compound also strives for higher medicinal status,
undergoing dozens of clinical trials for various ailments, from osteoporosis to cancer 3, 5ꢀ7
Generally, the sources of
are threefold: soy processing8ꢀ10, biotechnology11ꢀ13 and chemical
synthesis14. The most complicated situation is in the field of medical use of genistein
.
1
containing materials derived from the first two sources. Soybean processing provides several
categories of products containing isoflavones, e.g., soy protein concentrates or isolates (SPC
and SPI, respectively), in which the content and quantitative composition depends on method
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of extraction and isolation. As a result, genistein available in supplements is not the pure
substance, but only a component of complex matrix of biological origin, which contains a
mixture of isoflavones. In many countries soy isoflavone extracts and supplements are
available without a prescription (as dietary supplements) and such preparations are often not
standardized. For example, the results described by Chua et al15 revealed a lack of uniformity
among the products tested and the significant variability in the composition of the same
formulation over time. Moreover, the majority (67%) of the products contained less than 90%
of the label amounts. The use of plantꢀderived genistein for medical applications could be
especially challenging for dosage development (dose should be consistent, clinically relevant
and safe) and interpretation of research study results. In some cases16 the whole extract is
claimed to provide better biological effects than the mixture of the individual components.
Therefore, unlike diet supplementation use, clinical trials should be performed on high purity
certified API material, which may be available more conveniently as a result of the chemical
synthesis. However samples obtained in academic laboratories performed according to
published literature procedures are also unacceptable for such purpose, based on current
pharmaceutical law, which requires a specified and certified material from validated technical
process for qualified biological testing.
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Genistein, first isolated from dyers broom (Genista tinctoria) at the end of 19th
century17, with structure elucidated in 192518 soon became a synthetic target in connection
with disclosed oestrogenic activity 18, but such efforts intensified considerably towards the
end of 20th century, after it became evident that
1
is an efficient inhibitor of tyrosine kinases19
and can serve in this capacity as a multipurpose biochemical and pharmacological probe20,
with realistic perspective for a drug candidate position. Numerous synthetic approaches to
naturally occurring isoflavones (and their analogs) have been carried out14, 21ꢀ23. Evaluation of
the subject literature can be summarized as follows: i) there are basically four main chemical
synthetic approaches by which isoflavones can be obtained: chalcone pathway, deoxybenzoin
pathway, 3ꢀhalogene chromenone arylation and flavanone isomerisation (Scheme 1); ii) for all
listed approaches numerous experimental variants have been elaborated, since particular
patterns of substitution and types of protective groups used required modification of a
standard procedure; iii) to our knowledge, there are no reports in scientific literature on a
scale up and process development, involving any of the numerous laboratory procedures14.
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R3
OH
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R2
R2
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R3
O
O
a
b
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O
O
R1
R2
R3
(HO)2 B
R3
c
d
R3
O
R1
X
O
O
R2
R2
R1
X = halogen
O
Scheme 1. Four main chemical synthetic approaches for obtaining isoflavones: a. chalcone
pathway, b. deoxybenzoin pathway, c. 3ꢀhalogene chromenone arylation, d. flavanone
isomerisation
Considering all technical and operational complexities along the way, we have decided to go
for deoxybenzoin option as a platform for method and process development. The innovative
process for manufacturing of
1 using deoxybenzoin approach has been outlined by
Grynkiewicz24 and is covered by patents25ꢀ27. Deoxybenzoin route was selected from many
available synthetic options because its operational simplicity – no protection of phenolic
moieties is required during the entire process.
Despite the relative simplicity and good results (yield, purity of the product) in kilogram
scale, the initially designed process24ꢀ27 had some drawbacks and limitations. The former
route (see Fig. 5a) required the use of large amounts of such inconvenient and corrosive
materials as HClgas or 1,2ꢀdimethoxyethane (DME). Long total reaction time of the first step
and large amount of waste produced can also be indicated as additional disadvantage.
According to the Green Chemistry rules28, 29 and EU regulations30, the production processes
should be designed in a way that minimize the use and generation of hazardous substances, as
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well as to reduce the waste, energy and costs. Therefore, in order to meet new standards of
green chemistry, as well as the demands and requirements of the pharmaceutical market in
relation to the synthetic equivalents of active substances obtained from natural sources, we
decided to optimise the initially developed process of
1 synthesis.
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RESULTS AND DISCUSSION
In the first step of the former process (Scheme 2, Step A and A.1, A.2 in Fig. 5a), the
intermediate ꢀ 2,4,6,4’–tetrahydroxydeoxybenzoin ( ) ꢀ was formed in the HoubenꢀHoesh
reaction1, 31, 32, as a result of standard electrophilic substitution of unprotected phloroglucinol
) with 4ꢀhydroxyphenylacetonitrile ( ). The reaction was carried out in an ether solvent
previously saturated with dry hydrogen chloride (HClgas, 16 fold molar excess with respect to
the molar equivalent of ), using zinc chloride (ZnCl2) as a catalyst. The obtained
intermediate was further cyclized into , following formylation which is indispensible for
3
(6
5
6
3
1
the chromenone ring closure. Generally, for formylation there is a choice of reaction
conditions between Vilsmeierꢀlike, FriedelꢀCrafts like, or Cꢀalkylation of enolizable ketone
type. However, in all cases rather large excess of a formylation reagent is needed in order to
attain reasonable yield of the cyclized product. Considering a corrosive environment of the
first two variants, alkaline conditions for the Cꢀformylation looked more operationally facile.
Since most of such reactions were performed in heterogeneous mixtures, with use of
inorganic bases in organic solvents, our attention was drawn to another possibility – mixed
formic anhydrides in the presence of tertiary amines33 (Scheme 2, Step B and B.1, B.2, B.3 in
Fig. 5a).
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Scheme 2. The process of genistein synthesis; former process conditions according to
Achmatowicz et al27 (see also Fig. 5a)
OPTIMISATION OF THE FIRST STEP OF THE PROCESS
As it was mentioned above, in the first step of the former process27 3 was obtained in the
reaction of commercially available substrates:
HClgas, and with anhydrous ZnCl2 as a catalyst. The gaseous HCl was used in the 16ꢀfold
excess in relation to , overall reaction time of step 1 was 96 hours and the process was
6 and 5, in DME previously saturated with
6
conducted in 0ꢀ5ºC. Through the reaction, resulting intermediate (ketimine hydrochloride
precipitated from the reaction mixture, and next, after filtration, was hydrolyzed in the
presence of diluted HClaq.
4)
In the first step of our studies verification of the previously27 described procedure was
conducted, considering limited stability of the ether solvent in the presence of HCl/ZnCl2.
Several experiments in a small scale (from 0.4 g to 21.0 g of
6, see 1.1ꢀ1.8 in Table 1) were
performed, to establish the optimal quantity of HClgas, catalyst and solvent. The obtained
results (examples 1.2, 1.3 and 1.6ꢀ1.8) revealed, that the reaction run in a satisfactory way and
in shorter period of time, with the use of much smaller amounts of HClgas and room
temperature. At that stage of the study it was found that the optimal amount of gas was 6
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equiv. in relation to
6, which corresponds to 37.5% of the initial quantity (for detailed
4
5
comparison of former27 and novel process see Supporting data). For the catalyst, it was
6
7
confirmed that previously established amount (0.5 equiv.) was optimal. The decrease of the
amount of ZnCl2 resulted in lowering the yield of
4 (Table 1, exp. 1.1). On the other hand,
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increasing the amount of catalyst did not translate into an improvement of the yield of
(Table 1, exp. 1.3).
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Table 1. Conditions and results of the
3
synthesis (step A)a
6,d
mol/L
T,
°C
Time,
h
Purity of
Molar ratiosc
Yield, %
3,e%
Experiment Batch size,b
No.
kg
ZnCl2
HClgas
4
3
1.1
1.2
4.2·10ꢀ4
4.2·10ꢀ4
4.2·10ꢀ4
4.2·10ꢀ4
6.3·10ꢀ4
2.2·10ꢀ3
2.1·10ꢀ2
2.1·10ꢀ2
7.9·10ꢀ1
7.9·10ꢀ1
7.9·10ꢀ1
7.9·10ꢀ1
3.0·100
3.6·101
3.6·101
3.6·101
0.2
0.5
0.8
0.9
0.0
0.6
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
0.5
6.0
6.0
6.0
3.0
1.4
5.2
6.1
6.1
6.1
6.1
4.4
3.9
4.7
5.2
5.0
4.6
0.6
0.6
0.6
0.6
1.0
0.6
2.7
3.3
0.8
0.9
0.9
0.9
0.9
0.9
0.9
0.9
rt
rt
24
24
24
24
48
23
24
24
51
30
46
27
29
32
32
30
30
51
45
ꢀg
ꢀ f
ꢀ f
ꢀ f
ꢀ f
ꢀ f
ꢀ f
ꢀ f
ꢀ f
ꢀ f
ꢀ f
1.3
rt
1.4
1.5h
rt
rt
rti
rtk
2.5
ꢀ j
1.6
45
82
63
56
74
75
81
80
79
83
79
71.3
98.0
n.d.
81.0 l
97.8
95.4
95.7
99.0
97.5
97.6
97.6
1.7
92
66
77
84
77
92
82
ꢀ m
ꢀ m
1.8
rt
1.9
17ꢀ20
19ꢀ21
19ꢀ25
25ꢀ28
19ꢀ22
17ꢀ22
17ꢀ22
17ꢀ22
1.10
1.11
1.12
1.13
1.14
1.15
1.16
ꢀ m
a
b
c
General process conditions: see Scheme 2; batch size related to amount of 6; Molar ratios related to molar
d
e
equivalent of 6, (molar ratio of 6:5 1:1.14); initial concentration of 6 in pure DME; determined by means of
f
g
HPLC (area %); processes terminated at the stage of 4 synthesis; no precipitate present, conversion of 6
determined by means on 1H NMR (91.6%); h molar ratio of 6:5 1:0.77; i initial period of 3 hours carried out at 0ꢀ
5°C; j process carried out directly to formation of 3, without isolation of 4; initial period of 7 hours carried out
k
at 0 ꢀ 10 °C; l shortened hydrolysis; m yield not determined due to residual solvent present in 4.
The next stage of the process upgrade involved the gradual scaling up of Step A.2 (Table 1,
exp. 1.9ꢀ1.16). Various process parameters were reꢀdefined, in a way to lead the reaction
without initial cooling. Applying temperature of 17ꢀ28 ºC and cutting the reaction time to 27ꢀ
32 hours, allow to obtain
3 of suitable purity (95.7%ꢀ99%, according to HPLC) and with
acceptable yields (79%ꢀ83%, see exp. 1.12ꢀ1.16, Table 2). Using of ~3.9 and 4.4ꢀfold excess
of HClgas gave products with a satisfactory yield, but having a slightly lower purity (see Table
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1, exp. 1.11 and 1.12). During the further scale up (to the planned size of the batch, 50 kg) the
amount of HClgas was ultimately reduced to 5 equivalents (that states only 31% of the initial
quantity ꢀ 5 instead of 16 equivalents). At the same time, DME quantity was decreased by
~50%. With no negative influence on quality of
3
, all these actions led to a threeꢀfold
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reduction in acidic waste. The purity of the obtained
3, according to HPLC method, was very
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good (above 97%, exp. 1.14ꢀ1.16 in Table 1). In all mentioned experiments the only impurity
higher than 0.5% was that with RRT=0.40.
The introduced important technological modification consisted in direct saturation of the
reaction mixture with HClgas, instead of charging HCl as a solution in DME. This led to the
simplicity and safety of the reaction and improved the economy of the process (by reducing
the reaction time). The elimination of the step of solvent saturation allowed also to eliminate
the difficulties associated with the loading of the other reactants. Elongation of
reaction time (6 instead of 3.5 hours) resulted in almost complete conversion of this
compound into desired intermediate 3. For the complete and rapid hydrolysis of as well as
4 hydrolysis
4
for the homogeneity of the suspension (reaction mixture) fast agitation (80rpm) and methanol
addition (4.5%) are necessary.
OPTIMISATION OF THE SECOND STEP OF THE PROCESS
The second step of the process (Scheme 2, Step B) included formation of
reaction of with mixed acetoformic anhydride and in the presence of Et3N. According to the
former procedure27, intermediate
was isolated, hydrolyzed under alkaline conditions, and
1, by the
3
2
the reaction mixture was neutralized by the concentrated HClaq.
Environmental and economic aspects of the process were particularly considered when
optimizing this stage, including reduction of consumption of hazardous reagents (to diminish
of the amount of difficult waste produced) and of energy. The overall reꢀadjustment of reagent
input can be summarized as follows: DME was eliminated from the reaction medium, which
removed danger of acid catalysed ether bond fission as a side reaction. Concomitantly, excess
of acetic anhydride and formic acid was slightly increased (from 3.3 to 4.3 equiv. relative to
3
). The amount of Et3N was reduced three times (from 6 to 2 equivalents). Addition of Et3N
into the reaction mixture was carried out in a room temperature, instead of cooling the
mixture to 5ꢀ10 ºC. And finally, the number of operations has been reduced by omitting the
intermediate
2 isolation.
Purity of and yield aspects were also considered. The preliminary degradation study of
1
genistein showed, that the substance is unstable under basic conditions. Moreover, careful
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analysis of several experiments of hydrolysis of
2
showed, that alkaline conditions promotes
4
5
the formation of difficult to remove impurities. Therefore, in the new procedure, directly after
6
7
adding Et3N, the produced intermediate
2 is hydrolyzed (without isolation) under acidic ꢀ
instead of basic ꢀ conditions, by adding of HClaq dropwise to the reaction mixture. Several
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experiments in various scale were performed (see Table 2) and as a result crude
obtained in a form of hemihydrates.
1 were
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Table 2. Conditions and results on the synthesis of
1
(step B)a
3 applied
Crude 1
Crystallized 1
Time of
reaction
with Et3N,
h
Imp
RRT 1.07, b
%
Imp
Batch size, Purity,b
Yield, Purity, b
Yield Purity, b
No.
RRT 1.07, b
%
kg
%
%
%
%
%
2.1
2.2
2.3
2.4
2.5
2.6
2.7
2.8
2.9
2.10
4.0·101
3.0·101
4.0·101
2.0·10ꢀ2
2.0·10ꢀ2
2.0·10ꢀ2
5.0·10ꢀ1
5.0·10ꢀ1
2.0·10ꢀ2
1.0·10ꢀ1
97.48
97.60
97.61
97.80
97.80
97.80
97.80
97.80
99.19
99.19
20
20
21
43
20
22
18
48
22
22
76
71
80
86
90
94
69
90
90
94
99.79
99.84
99.82
99.61
99.60
99.86
ꢀd
0.05
0.00
0.09
0.12
0.07
0.05
ꢀ d
89
94
89
ꢀc
99.94
99.94
99.93
ꢀ c
0.06
0.06
0.07
ꢀ c
79
75
ꢀc
99.83
99.93
ꢀ c
0.11
< 0.05
ꢀ c
99.18
99.76
99.81
0.19
0.05
0.00
ꢀc
ꢀ c
ꢀ c
74
74
99.89
< 0.05
< 0.05
99.90
c
a
b
General process conditions: see Scheme 2, step B; determined by means of HPLC (area %); batch of 1 was
not crystallized, d purity was not determined.
It should be noted, that excess of such formylation reagents as acetic anhydride and formic
acid is bound to be used up by the phenolic groups present, resulting in formation of Oꢀ
formates, and to certain extend also Oꢀacetates. It can be assumed that the formate esters are
very unstable and hydrolyse upon contact with aqueous solution. Phenolic acetates on the
other hand are less susceptible to hydrolysis, and that is why the reaction mixture is treated at
the end with alkaline solution. A competition between formylation and acetylation upon
application of mixed anhydride apparently takes place also during Cꢀacylation reaction.
Consequently, very small but isolable amount of genistein analog bearing Cꢀ2 methyl group
could be identified in a crude product of synthesis. This impurity, identified in crude
1
samples using LCꢀMS/MS method (see Table 2, Imp RRT 1.07), was the most problematic.
Even though its content was only slightly higher of the acceptable limit (0.10%), it was
virtually impossible to remove this compound during cleaning processes. The only solution to
prevent the formation of this impurity was elaboration of the reaction conditions promoting
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formylation. The effect of reaction time and purity of the used intermediate 3 on yield and
purity of 1 was examined. It was found, that mixing time with Et3N is a critical parameter
because it has a great impact on the quality of the resulting product. Optimal reaction time
with Et3N was determined to be 20ꢀ22 hours. Shortening of the reaction time resulted in
lowering the yield of the crude 6 (exp. 2.7, Table 2), whereas doubling it had no influence on
yield, but led to the formation of undesirable impurity at a level exceeding the limit value (see
exp. 2.4 and 2.8 in Table 2). It was also found, that impurity profile of the used 3 had little
effect on the crude 1 (genistein hemihydrate) quality.
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Solid state form (i.e. the crystalline or amorphous form) of a drug candidate can be critical to
its pharmacological properties and to its development as a viable API34,35. Therefore, the last
operation of the step B (step B.3) was crystallization of crude 1 (genistein hemihydrate; see
the last part of the Experimental section), from anhydrous ethanol, in order to obtain a desired
crystal structure (marked as “genistein after crystallization”, see Figure 2 and 3). The yield of
this process was in the range of 70% to 82% and the final product met requirements for API36,
37. Because 1 is a hydrophobic compound (class II compounds in the biopharmaceutical
classification system with low solubility and high permeability38), reduction of the particle
size by the micronization process was additionally performed to increase solubility and
bioavailability of this API. However, as it was proven, this process did not influence the
crystal structure of 1 (see comparison of XRPD profiles on Figure 4).
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Figure 2 Comparison of DSC curves of samples: crude 1 (hemihydrate) and the same batch
4
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Figure 3 Comparison of XRPD profiles of samples: crude 1 (hemihydrate) and the same batch
crystallized from anhydrous ethanol (the required form)
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Figure 4 Comparison of theoretical powder pattern39 of 1 with the experimental patterns after
crystallization and micronization of 1
RESULTS OF OPTIMISATION OF THE GENISTEIN MANUFACTURING PROCESS
The proposed changes of reactions conditions of both stages led to a significant
improvement of the economics of the process. The amount of HClgas used in step A was
reduced to the 31% of the initial quantity (5 instead of 16 equivalents) and gaseous HCl was
bubbled directly into the reaction mixture instead of charging HCl as a solution in DME. In
addition, the amount of solvent was reduced by almost 50%. Revision of reaction parameters
allowed the formation of intermediate 4 at room temperature and without the initial cooling.
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Moreover, the overall reaction time of step A was considerably shortened. All these
modifications allowed to perform the reaction effectively at pilotꢀplant scale, utilize of the
laboratory equipment in a more efficient manner and minimize the problem of neutralization
the large quantities of HCl solution.
9
For the step B of this process the amount of reagents used (especially Et3N) was reduced,
DME ꢀ flammable and toxic, class 2 solvent that should be limited in pharmaceutical
products40 – was eliminated, filtration of the intermediate 2 was omitted (reducing the number
of operations, shortening of the total duration time of step B). To enhance the efficiency of the
hydrolysis reaction of 2 and to avoid degradation of the resulting 1 (step B), the reaction was
carried out in an acidic environment (switch from alkaline conditions in the former
procedure).
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Comparison of the amounts of the reagents used in the synthesis of 1 before and after
optimisation of the process is illustrated by the Sankey diagram (Figure 5) and in Figure 6.
Figure 5. Sankey diagram: comparison of former (a) and novel (b) technology of genistein
manufacturing
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Batch load, kg
0
10000 20000 30000 40000 50000 60000 70000 80000 90000 100000
6
7
8
Water
Ethanol
9
DME
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Methanol
HCl
Triethylamine
Acetic anhydride
NaOH
4ꢀHydroxyphenylacetonitrile
Floroglucinol
Formic acid
Zinc chloride
Activated carbon
Former technology
Novel technology
Figure 6 Batch loads in novel and former genistein technologies
CONCLUSIONS
Guidelines of Green Chemistry and Engineering are important for process design and
development especially in pharmaceutical industry. REACH30 places greater responsibility on
industry to manage the risks that chemicals may pose to the health and the environment.
Therefore synthetic methods should be designed to use and generate substances that possess
little or no toxicity to human health and the environment and compounds of high concern
should be replaced by less dangerous equivalents. In accordance with the green chemistry
rules, new conditions for genistein manufacturing have been proposed. Currently optimised
method, in addition to the obvious advantages associated with the reduction or complete
removal of harmful and difficult to handle reagents (HCl, DME, Et3N), shortening the
duration time of most of the steps and thus a reduction in energy consumption, reduction of
number of operation steps, simplifying the technology (saturating the reaction mixture with
HClgas) and a significant decrease of produced waste, offers facile transition from laboratory
to pilot plant scale. This applies to the scaling up of both steps: intermediate 3 formation as
well as transformation of this compound into 1. Both productivity and quality of the
compounds obtained were reproducible, which demonstrates the detailed and careful
examination of the performed reactions. It should be underlined, that intended switch of
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genistein molecule status from an expensive reagent to a specialty chemical was achieved as
planned. The optimised manufacturing process as well as all analytical methods used for
assessing of quality of produced 1 were validated. The obtained batches were certified
according to the inꢀhouse specification including tests of identity (including polymorphic
form), purity, assay, total ash, content of water and residual solvents, as well as the study of
particle size distribution. The proposed technology assures a high yield and purity of genistein
that meet the requirements for pharmaceutical substances.
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EXPERIMENTAL SECTION
GENERAL PROCEDURES
Starting materials: 6 and 5 were purchased from Allorachem SRL (Rimini, Italy). According
to the supplier statement, substances used in the synthesis of both compounds meet the
requirements of ICH Q7 guideline41 of the European Medicines Agency for the development
of the active substances. 5, purchased from Allorachem SRL, was however manufactured by
Chizhou Feihaoda Chemical Co. (Chizhou, China), in a well documented twoꢀstep process.
Regardless of the supplier data, 5 was tested and certified in PRI based on the inꢀhouse
specification (purity 99.7% and assay 99.3% according to HPLC methods, confirmed
identity). Testing and certification was also performed for 1 (assay 99.5%, confirmed
identity), manufactured by Yancheng Huahong Chemical Co. (Yancheng, China). Reagents
and solvents were obtained from commercial sources and were used as received.
1H NMR (200 MHz) and 13C NMR (50 MHz) spectra were recorded using a Varian Gemini
2000 spectrometer. The 1H and 13C chemical shift data were reported as
δ (ppm) using solvent
as internal standard (3.30 ppm and 49.0 ppm for methanol and 1.73 ppm and 25.3 ppm for
THF, respectively). Infrared (IR) spectra were recorded using Perkin Elmer instrument. HPLC
analysis were performed on Waters and Shimadzu systems, using Luna C18 (Phenomenex)
250 mm x 4.6 mm, 5 µm column.
SCALE UP SYNTHESIS
2,4,4’,6–tetrahydroxydeoxybenzoin (3, CAS: 15485-65-1)
In a 1000 L enamel reactor with fixed parameters of mixing, equipped with anchor stirrer, 1,2ꢀ
dimethoxyetane (264.2 kg) was placed, followed by 6 (36.0 kg ; 285.5 mol), 5 (43.2 kg ;
324.4 mol) and anhydrous ZnCl2 (18.9 kg; 138.6 mol), and the mixture was stirred at room
temperature until substrates dissolved. Then HClgas was added slowly, until 47.5 kg (1301.4
mol) of the reagent has been absorbed. The obtained solution was stirred at RT for 48 hrs,
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then the reactor content was cooled to 10oC, water (14.0 kg) was added and stirring was
continued for another 2.5 hrs, while temperature was kept below 10oC. The yellowꢀorange
solid of ketoimine hydrochloride 4 was filtered off on pressure filter, washed with 25.2 kg
DME and 43.2 kg of water. The solid (99 kg), without drying, was placed in a 1000 L reactor,
to which water (388.8 kg), concentrated aqueous hydrochloric acid (29.5 kg) and methanol
(14.6 kg) were added and the mixture was stirred under reflux until 4 was exhausted (TLC
control, approx. 12 hrs.). Then, the mixture was cooled down to 10oC, stirred for 2 hrs., and
precipitate was filtered of vacuum filter, and washed with water (47.9 kg) twice. The solid
was dried in fluid dryer at 60ꢀ70ºC for 1 hr. 3 (59.1 kg; 79.5 % yield) thus obtained featured
over 97% purity, according to HPLC (% area). The intermediate was applied in the next step
of synthesis without further purification.
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IR (KBr, cmꢀ1 ; Inter alia): 3492, 3327, 3208, 1646, 1611, 1591, 1540, 1515, 1475, 1417,
1358, 1304, 1214, 1078, 993, 818, 798, 529.
1HꢀNMR (200 MHz, CD3OD), δ (ppm): 4.26 (s, 2H); 5.80 (s, 2H); 6.69 (d, J = 8.6 Hz, 2H);
7.05 (d, J = 8.6 Hz, 2H).
13CꢀNMR (CD3OD), δ (ppm): 95.73 (2xC); 105.21; 115.93 (2xC); 128.13; 131.71 (2xC);
156.86; 165.84; 166.25; 205.07 (C=O)
Genistein (1, CAS: 446-72-0; batch No. 030314)
In a 600 L reactor acetic anhydride (67 kg; 656.3 mol) was placed, and formic acid (98%,
29.3 kg ; 636.6 mol) was added while stirring, with temperature maintained below 35oC. After
addition was completed, the mixture was cooled to 20oC, 3 (40.0 kg; 153.7 mol) was added
and the mixture was stirred for one hour at RT. Next, Et3N was added dropwise, while
maintaining temperature below 20ºC. Stirring was continued for 24 hrs., then concentrated
aqueous hydrochloric acid (47.2 kg) was dropped in, at temperature below 22ºC. After next
22 hrs at ambient temperature, water (30.0 kg) was added and stirring was continued for the
next hour. Then aqueous solution of sodium hydroxide (8.0 kg of NaOH in 40 kg of water)
was dropped in while stirring, maintaining the temperature below 30ºC. After addition was
completed, the precipitation formed was centrifuged (1000 rpm, diameter 1000 mm) washed
with water twice (40.0 kg) and centrifuged again. The solid discarded from centrifuge was
transferred to the same 600 L reactor containing methanol (168.0 kg) and stirred, then sodium
hydrogen solution (10.85 kg NaOH in 188.0 kg of water) was added slowly, at temperature
below 22ºC. To the reaction mixture aqueous HCl was added (29.0 kg of concentrated
hydrochloric acid in 19.6 kg of water) in temperature below 25ºC and the suspension was
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stirred for the next hour at ambient temperature. The obtained solid was centrifuged (1000
rpm, diameter 1000 mm), washed with water (168.0 kg), centrifuged again and dried (drying
air chilling) under 55ºC for 48 hrs. The crude 1 was obtained in a form of hemihydrate (batch
020614; 59.1 kg; 79.5 % yield; purity 99.81 % according to HPLC).
9
Pharmaceutical quality 1 in a required crystal form was obtained after crystallization from
ethanol. Crude 1 (20.0 kg), obtained as described above, was placed in a 630 L reactor,
followed by anhydrous 99.98% ethanol (420.0 kg). The mixture was brought to boiling (1
dissolution), then filtered through pressure filter (cotton type BT 17) while hot. The filtrate
was concentrated to the volume of 100 L, under diminished pressure, at 45 – 60ºC. After
cooling the residue to the ambient temperature, water was added (90.0 kg) while stirring and
after 5 hrs the obtained solid was centrifuged, washed with water (40.0 kg) and dried on air at
40 – 55ºC. Genistein (18 kg ; crystallization yield ca. 90 %) was obtained as substance of
pharmaceutical quality (99.90%, area % by HPLC; Imp RRT 1.07 < 0.1%).
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IR (KBr, cmꢀ1 ; inter alia): 3411, 3104, 1651, 1615, 1570, 1519, 1504, 1424, 1361, 1309,
1202, 1179, 1145, 1043, 911, 840, 790.
1HꢀNMR (200 MHz, THFꢀd8), δ (ppm): 6.17 (d, J = 2,2 Hz, 1H); 6.26 (d, J = 2,2 Hz, 1H);
6.78 (m, 2H); 7.41 (m, 2H); 8.02 (s, 1H); 8.50 (bs, 1H); 9.34 (bs, 1H); 13.02 (s, 1H).
13C NMR (THFꢀd8), δ (ppm): 94.13; 99.73; 106.20; 115.82; 122.95; 124.17; 130.84; 153.78;
158.73; 159.08; 164.24; 165.16; 181.46.
Acknowledgements
The study was funded by The National Centre for Research and Development under the
Applied Research Programme (path B), fund No. PBS1/B7/7/2012.
Supporting data
Supporting data file contains: (1) tabular version of Sankey chart; (2) the synthetic scheme
and the statement of compliance with the ICH Q7 guideline provided together with compound
2 (4ꢀhydroxyphenylacetonitrile, now renumbered as 5) by Allorachem company.
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(41)ICH Guideline Q7, Good Manufacturing Practice Guide for Active Pharmaceutical
Ingredients; International Conference on Harmonization, 2000.
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ACS Paragon Plus Environment